A kit for detecting and analyzing the non-free nucleoprotein content of rabies vaccine and / or detecting the integrity of rabies virus particles

Abstract

The present invention discloses a kit for detecting and analyzing the content of non-free nucleoprotein in rabies vaccine and / or detecting the integrity of rabies virus particles. The kit contains a monoclonal antibody against rabies virus glycoprotein and a monoclonal antibody against rabies virus nucleoprotein. The kit can specifically detect the non-free nucleoprotein in rabies vaccine samples and can obtain a standard curve with a strong correlation, thereby achieving the purpose of quickly obtaining the integrity of virus particles.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine detection, in particular to a kit for detecting and analyzing the non-free nucleoprotein content of rabies vaccine and / or detecting the integrity of rabies virus particles. Background Art

[0002] Rabies is mainly caused by infection with the rabies virus (RV). It usually occurs after being scratched or bitten by animals such as cats and dogs. Patients often show symptoms such as hydrophobia, pharyngeal muscle spasm and fear of wind. There is currently no specific drug that can effectively treat rabies in clinical practice, so once the disease occurs, the mortality rate of patients is almost 100%. The RV genome consists of 11928 to 11932 nucleotides, which is a single-stranded, non-segmented negative-strand RNA with 5 large open reading frames encoding 5 structural proteins. The order of the genome from the 3' end to the 5' end is N, P, M, G and L genes, which encode viral nucleoprotein (N), phosphoprotein (P), membrane matrix protein (M), glycoprotein (G) and transcription large protein (L). In recent years, with the increasing number of pets, healthy animals still have a virus-carrying rate of about 15%. In order to effectively prevent rabies, the best way at this stage is to get a rabies vaccine injection.

[0003] During the development and production of rabies vaccines, the integrity of rabies virus particles needs to be tested. The integrity of virus particles is a key indicator for evaluating and ensuring the quality of vaccines. Currently, electron microscopy observation and naked eye evaluation of virus particle integrity are still the main methods. However, the electron microscopy sample preparation process is relatively complicated, with high requirements for instruments and equipment, and the sampling and processing methods have a great impact on the experimental results, making the electron microscopy detection method difficult to implement and poor in repeatability.

[0004] There is an urgent need to develop a simple and effective test method that can be performed in vaccine quality control-related testing laboratories to evaluate the viral particle integrity of rabies vaccines. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a kit for detecting and analyzing the non-free nucleoprotein content of rabies vaccine and / or detecting the integrity of rabies virus particles. Because glycoprotein is the only protein exposed on the surface of the rabies virus envelope. If the monoclonal antibody against glycoprotein is coated on the solid surface, the coated anti-glycoprotein monoclonal antibody will specifically bind to the glycoprotein on the surface of the rabies virus and then capture the virus particles. At this time, if the virus particles are intact, the nucleoprotein cannot be recognized and detected by the labeled nucleoprotein antibody because it is wrapped inside the virus particles. When the captured rabies virus particles are incomplete, the nucleoprotein in the non-free state in the virus particles will be exposed and recognized by the labeled nucleoprotein antibody and detected. Using this detection mode of non-free nucleoprotein, the closer the rabies virus particles in the vaccine sample are to the complete state, the lower the detection signal. Within a certain range, the detection signal of non-free nucleoprotein increases as the integrity of the particles decreases, and then as the rabies virus particles are further severely damaged, the nucleoprotein detaches from the particles and becomes free and cannot be detected, resulting in a decrease in the signal. Therefore, this detection mode can be used to easily evaluate and monitor the integrity of viral particles.

[0006] The first object of the present invention is to provide a hybridoma cell line that secretes monoclonal antibodies against rabies virus glycoprotein.

[0007] The second object of the present invention is to provide the anti-rabies virus glycoprotein monoclonal antibody secreted by the hybridoma cell line.

[0008] The third object of the present invention is to provide a hybridoma cell line that secretes monoclonal antibodies against rabies virus nucleoprotein.

[0009] The fourth object of the present invention is to provide a monoclonal antibody against rabies virus nucleoprotein secreted by the hybridoma cell line.

[0010] The fifth object of the present invention is to provide the use of any one or two of the hybridoma cells and / or any one or two of the monoclonal antibodies in the preparation of a kit for detecting the non-free nucleoprotein content of rabies virus and / or rabies vaccine and / or detecting the integrity of rabies virus particles.

[0011] The sixth object of the present invention is to provide an immunoassay kit for detecting the content of non-free nucleoprotein of rabies virus and / or rabies vaccine, and / or the integrity of rabies virus particles.

[0012] The seventh object of the present invention is to provide a method for detecting the non-free nucleoprotein content of rabies virus and / or rabies vaccine, and / or the integrity of rabies virus particles.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

[0014] The present invention provides a hybridoma cell line YB-RV22 that secretes a monoclonal antibody against rabies virus glycoprotein, which is deposited in the China Center for Type Culture Collection, with a deposit address of Wuhan University, Wuhan, China, a deposit number of CCTCCNO: C202220, and a deposit date of January 19, 2022.

[0015] And, the anti-rabies virus glycoprotein monoclonal antibody YB-RV22 secreted by the hybridoma cell line.

[0016] The anti-rabies virus glycoprotein monoclonal antibody YB-RV22 is of IgG1 type, and its light chain is a kappa chain.

[0017] The present invention also provides a hybridoma cell line YB-RV26 that secretes monoclonal antibodies against rabies virus nucleoprotein, which is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, the deposit number is CCTCCNO: C2022283, and the deposit date is: September 21, 2022.

[0018] And, the hybridoma cell line secretes the monoclonal antibody YB-RV26 against rabies virus nucleoprotein.

[0019] The anti-rabies virus glycoprotein monoclonal antibody YB-RV26 is of IgG1 type, and its light chain is a kappa chain.

[0020] It is also claimed to protect the use of any one or two of the hybridoma cells and / or any one or two of the monoclonal antibodies in the preparation of a kit for detecting the content of non-free nucleoprotein of rabies virus and / or rabies vaccine and / or detecting the integrity of rabies virus particles.

[0021] An immunoassay kit for detecting the content of non-free nucleoprotein of rabies virus and / or rabies vaccine, and / or the integrity of rabies virus particles, contains one or two of the monoclonal antibodies.

[0022] Preferably, the immunoassay kit is a time-resolved fluorescent immunoassay kit, using the monoclonal antibody against rabies virus glycoprotein as the primary antibody; and the monoclonal antibody against rabies virus nucleoprotein labeled with a lanthanide ion complex as the secondary antibody.

[0023] More preferably, the lanthanide ion is Eu 3+ 、Dy 3+ , Tb 3+ 、Sm 3+ One or more of .

[0024] Further preferably, the lanthanide ion is Eu 3+ .

[0025] Preferably, the lanthanide ion complex is N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (SCN-Ph-DTTA)-Eu 3+ (SCN-Ph-DTTA-Eu 3+ ).

[0026] More preferably, it further contains one or more of a blocking solution, an enhancement solution, a washing solution, a labeling solution, a sample diluent and / or a secondary antibody diluent.

[0027] More preferably, the blocking solution is Tris-HCl buffer containing BSA.

[0028] Further preferably, the blocking solution is a 30-70 mmol / L Tris-HCl buffer solution with a mass concentration of 1-1.5 wt % BSA.

[0029] Most preferably, the blocking solution is a 50 mmol / L Tris-HCl buffer solution with a mass concentration of 1 wt % BSA.

[0030] More preferably, the enhancement solution is an acetic acid-phthalic acid buffer solution containing 40-60 μmol / L TOPO, 10-20 μmol / L β-NAT, 0.1-0.2 vol% Triton X-100, pH=7.5-8.0, and a concentration of 80-100 mmol / L.

[0031] More preferably, the enhancement solution is an acetic acid-phthalic acid buffer solution containing 50 μmol / L TOPO, 15 μmol / L β-NAT, 0.1 vol% Triton X-100, pH=7.8, and a concentration of 100 mmol / L.

[0032] More preferably, the washing solution is a Tris-HCl buffer containing 0.5-1wt% NaCl, 0.01-0.05wt% NaN3, 0.05-0.1vol% Tween-20, pH=7.5-8.0, and a concentration of 30-70mmol / L.

[0033] Further preferably, the washing solution is a Tris-HCl buffer containing 0.84 wt % NaCl, 0.01 wt % NaN3, 0.06 vol % Tween-20, pH = 7.8, and a concentration of 50 mmol / L.

[0034] More preferably, the labeling solution is a Na2CO3-NaHCO3 buffer solution containing 150-160 mmol / L NaCl, with a pH of 8.2-8.8 and a concentration of 40-60 mmol / L.

[0035] Further preferably, the labeling solution is a Na2CO3-NaHCO3 buffer solution containing 155 mmol / L NaCl, with a pH of 8.5 and a concentration of 50 mmol / L.

[0036] More preferably, the sample diluent is a Tris-HCl buffer containing 0.01% to 0.1wt% NaN3, 0.1% to 0.3wt% bovine serum albumin, a pH of 7.5 to 8.0, and a concentration of 40 to 60 mmol / L.

[0037] Further preferably, the sample diluent is a Tris-HCl buffer containing 0.05 wt % NaN 3 , 0.2 wt % bovine serum albumin, with a pH of 7.8 and a concentration of 50 mmol / L.

[0038] More preferably, the secondary antibody diluent contains 0.01-0.05wt% BSA, 0.01-0.1% Tween-20, 0.01-0.1wt% NaN3, pH=7.5-8.0, and Tris-HCl buffer with a concentration of 40-60mmol / L.

[0039] Further preferably, the secondary antibody diluent contains 0.03wt% BSA, 0.05vol% Tween-20, 0.05wt% NaN3, pH=7.8, and a Tris-HCl buffer with a concentration of 50mmol / L.

[0040] The present invention also claims a method for detecting the content of non-free nucleoprotein of rabies virus and / or rabies vaccine, and / or the integrity of rabies virus particles, comprising the following steps:

[0041] The anti-rabies virus glycoprotein monoclonal antibody is coated on an ELISA plate and blocked with a blocking solution to obtain a solid phase monoclonal antibody material;

[0042] The monoclonal antibody against rabies virus nucleoprotein is labeled with a lanthanide ion complex to prepare a secondary antibody;

[0043] The sample to be tested and the secondary antibody are added to the solid phase monoclonal antibody material, and then an enhancement solution is added for incubation to obtain an ELISA plate to be tested;

[0044] The ELISA plate to be tested is subjected to fluorescence measurement, and the fluorescence signal obtained from the sample to be tested is substituted into the standard curve equation obtained by fitting the calibration substance concentration and signal data to obtain the non-free nuclear protein content in the sample to be tested.

[0045] More preferably, the method comprises the following steps:

[0046] The anti-rabies virus glycoprotein monoclonal antibody is coated on an ELISA plate, washed with a washing solution, patted dry, then blocked with a blocking solution, washed with a washing solution, patted dry, to obtain a solid phase monoclonal antibody material;

[0047] The monoclonal antibody against rabies virus nucleoprotein, the labeling solution and the lanthanide ion complex are mixed, fully reacted, labeled and purified to prepare a secondary antibody;

[0048] The solid phase monoclonal antibody material is added with the sample to be tested and the secondary antibody, incubated, washed with a washing solution, and patted dry; then an enhancement solution is added for incubation to obtain an ELISA plate to be tested, wherein the sample to be tested is diluted with a sample diluent, and the secondary antibody is diluted with a diluent;

[0049] The ELISA plate to be tested is subjected to fluorescence measurement, and the obtained fluorescence signal is substituted into the standard curve equation obtained by fitting the calibration substance concentration and signal data to calculate the non-free nuclear protein content in the sample to be tested.

[0050] More preferably, the washing is washing the ELISA plate 4 to 6 times with 300 to 350 μL / well of washing solution.

[0051] More preferably, the ELISA plate is washed 6 times with 300 μL / well of washing solution.

[0052] More preferably, the purification is performed using a Sephadex G50 column.

[0053] More preferably, the blocking solution is a BSA solution.

[0054] More preferably, the blocking solution has a mass concentration of 1 to 1.5%.

[0055] Most preferably, the blocking solution has a mass concentration of 1%.

[0056] More preferably, the lanthanide ion is Eu 3+ 、Dy 3+ , Tb 3+ 、Sm 3+ One or more of .

[0057] Further preferably, the lanthanide ion is Eu 3+ .

[0058] More preferably, the enhancement solution is an acetic acid-phthalic acid buffer solution containing 40-60 μmol / L TOPO, 10-20 μmol / L β-NAT, 0.05-0.2 vol% Triton X-100, pH=7.5-8.0, and a concentration of 80-100 mmol / L.

[0059] More preferably, the enhancement solution is an acetic acid-phthalic acid buffer solution containing 50 μmol / L TOPO, 15 μmol / L β-NAT, 0.1 vol% Triton X-100, pH=7.8, and a concentration of 100 mmol / L.

[0060] More preferably, the washing solution is a Tris-HCl buffer containing 0.5-1wt% NaCl, 0.01-0.05wt% NaN3, 0.05-0.1vol% Tween-20, pH=7.5-8.0, and a concentration of 30-70mmol / L.

[0061] Further preferably, the Tris-HCl buffer contains 0.84wt% NaCl, 0.01wt% NaN3, 0.06vol% Tween-20, pH=7.8, and a concentration of 50mmol / L.

[0062] More preferably, the labeling solution is a Na2CO3-NaHCO3 buffer solution containing 150-160 mmol / L NaCl, with a pH of 8.2-8.8 and a concentration of 40-60 mmol / L.

[0063] Further preferably, the labeling solution is a Na2CO3-NaHCO3 buffer solution containing 155 mmol / L NaCl, with a pH of 8.5 and a concentration of 50 mmol / L.

[0064] More preferably, the sample diluent is a Tris-HCl buffer containing 0.01% to 0.1wt% NaN3, 0.1% to 0.3wt% bovine serum albumin, a pH of 7.5 to 8.0, and a concentration of 40 to 60 mmol / L.

[0065] More preferably, the buffer solution comprises 0.05 wt% NaN3, 0.2 wt% bovine serum albumin, a pH of 7.8 and a concentration of 50 mmol / L Tris-HCl.

[0066] More preferably, the secondary antibody diluent contains 0.01-0.05 wt% BSA, 0.01-0.1 vol% Tween-20, 0.01-0.1 wt% NaN3, and a Tris-HCl buffer having a pH of 7.5-8.0 and a concentration of 40-60 mmol / L.

[0067] Further preferably, the secondary antibody diluent contains 0.03wt% BSA, 0.05vol% Tween-20, 0.05wt% NaN3, pH=7.8, and a Tris-HCl buffer with a concentration of 50mmol / L.

[0068] Preferably, the lanthanide ion complex is a europium ion complex SCN-Ph-DTTA-Eu 3+ The weight ratio of the monoclonal antibody against rabies virus nucleoprotein is (0.1-0.3):1.

[0069] More preferably, the europium ion complex SCN-Ph-DTTA-Eu 3+ The weight ratio of the monoclonal antibody to the anti-rabies virus nucleoprotein is 0.2:1.

[0070] Preferably, the coating concentration of the primary antibody is 3-5 μg / mL; the use concentration of the secondary antibody is 1.8-2.2 μg / mL.

[0071] More preferably, the coating concentration of the primary antibody is 3 μg / mL; the use concentration of the secondary antibody is 2 μg / mL.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The present application utilizes anti-rabies virus glycoprotein monoclonal antibodies and anti-rabies virus nucleoprotein monoclonal antibodies to establish a detection method and a detection kit based on time-resolved fluorescence immunoassay, which can specifically detect non-free nucleoprotein in rabies vaccine samples and obtain a standard curve with a strong correlation, thereby achieving the purpose of quickly obtaining the integrity of virus particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is the western-blot identification result of anti-rabies virus glycoprotein monoclonal antibody YB-RV22.

[0075] Figure 2 This is the western-blot identification result of anti-rabies virus nucleoprotein monoclonal antibody YB-RV26.

[0076] Figure 3 Schematic diagram of the detection principle of time-resolved fluorescence immunoassay technology for non-free nucleoprotein of rabies vaccine.

[0077] Figure 4 This is the “concentration-fluorescence value” standard curve fitting diagram provided in Example 2 of the present application. DETAILED DESCRIPTION

[0078] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0079] Example 1 Preparation of anti-RV G protein monoclonal antibodies using hybridoma technology

[0080] 1. Preparation of immune agents:

[0081] Take 1 mL of the PM strain inactivated rabies virus solution with a concentration of 1 mg / mL provided by Guangzhou Baiyunshan Biological Products Co., Ltd. and mix it thoroughly with 1 mL of Freund's complete adjuvant as a basic immunizing agent; take 1 mL of the inactivated rabies virus solution with a concentration of 1 mg / mL and mix it thoroughly with 1 mL of Freund's incomplete adjuvant as a booster immunizing agent; wherein, Freund's complete adjuvant and incomplete adjuvant were purchased from Sigma.

[0082] 2. Immunization of animals:

[0083] Six 7-week-old female BLAB / c mice were selected for immunization. The first immunization: 0.1 mL of the basic immunization agent was injected subcutaneously on the back of the mouse; the second immunization: 14 days later, 0.1 mL of the booster immunization agent was injected subcutaneously on both sides of the mouse's groin; the third immunization: 14 days later, 0.1 mL of the booster immunization agent was injected subcutaneously on both sides of the mouse's shoulder and abdomen; the last immunization: 7 days later, blood was collected from the tail vein of the mouse, and the titer of the antibody in the serum was detected by indirect ELISA. When the antibody titer reached 1:10000, 0.1 mL of the booster immunization agent was injected into the mouse's abdominal cavity.

[0084] Specific method for detecting the titer of antibodies in serum by indirect ELISA method:

[0085] Inactivated rabies virus was used as the coating antigen, diluted to 1 μg / ml with a Tris-HCl buffer solution of pH=7.2 and a concentration of 50 mmol / L, added to the ELISA plate at 100 μL / well, coated overnight at 4°C and patted dry, and blocked with a 50 mmol / L Tris-HCl buffer solution of 1 wt% BSA in a mass volume ratio of 300 μL / well, blocked overnight at 4°C and patted dry for later use;

[0086] Mouse serum was diluted with Tris-HCl buffer containing 0.01% to 0.1wt% NaN3, 0.2wt% bovine serum albumin, pH 7.8, and 50mmol / L at 1:1000, 1:10000, and 1:50000 for later use, and 100 μL / well was added to the ELISA plate, incubated at 37°C for 60 min, washed with Tris-HCl buffer containing 0.84wt% NaCl, 0.01wt% NaN3, 0.06vol% Tween-20, pH=7.8, and 50mmol / L, and patted dry. Subsequently, 100 μL / well of HRP-labeled goat anti-mouse secondary antibody was added, incubated at 37°C for 60 min, washed and patted dry, and then 100 μL / well of TMB colorimetric solution was added, and the plate was incubated at 37°C in the dark for 10 min, and 50 μL / well of 1 mol / L The reaction was terminated with HCl and sent to BIO-Tek microplate reader for detection. The OD value was measured after the serum was diluted 1:10000 450 When the titer exceeds 1.5, the serum titer is considered to meet the fusion requirements. The serum titer results are shown in Table 1 below.

[0087] Table 1 Test results of mouse serum titer

[0088]

[0089] 3. Preparation of hybridoma cells:

[0090] 1. Experimental methods

[0091] Blood was collected 3 days after the last immunization, and the mouse spleen was obtained aseptically to prepare a spleen cell suspension; the mouse spleen cell suspension was fused with mouse myeloma SP2 / 0 cells at a ratio of 1:1 to form hybridoma cells. The cells were gently suspended in HAT culture medium (purchased from Sigma) and the cells were divided into 96-well plates at 200 μL per well; after three days of culture, the cell fusion was observed, the culture medium was replaced, and the culture was continued until clones were formed, and the culture medium was replaced seven days after fusion.

[0092] The cells producing anti-rabies virus monoclonal antibodies were screened by indirect ELISA method, and the positive wells with high titer were subcloned at least 3 times by limiting dilution method, and finally the positive hybridoma cells with a monoclonal cell line positive rate of 100% were obtained.

[0093] The specific method of using indirect ELISA is as follows: using inactivated rabies virus as the coating antigen, diluting it to 1 μg / ml with a Tris-HCl buffer solution of pH=7.2 and a concentration of 50 mmol / L, adding 100 μL / well to the ELISA plate, coating overnight at 4°C and patting dry, blocking with a Tris-HCl buffer solution of 50 mmol / L with a mass volume ratio of 1 wt% BSA, 300 μL / well, blocking overnight at 4°C and patting dry for later use;

[0094] The supernatant in the microplate after fusion culture was taken, and 100 μL / well was added to the ELISA plate, and incubated at 37°C for 60 min, and then washed with 50 mmol / L Tris-HCl buffer containing 0.84 wt% NaCl, 0.01 wt% NaN3, 0.06 vol% Tween-20, pH=7.8, and patted dry, and then 100 μL / well of HRP-labeled goat anti-mouse secondary antibody was added, and incubated at 37°C for 60 min, washed and patted dry, and then 100 μL / well of TMB colorimetric solution was added, and the plate was incubated at 37°C for 10 min in the dark, and 50 μL / well of 1 mol / L HCl was added to terminate the reaction and sent to BIO-Tek ELISA reader for detection;

[0095] The positive hybridoma cell lines detected by the ELISA method were inoculated into HT incomplete medium and subcloned at least 3 times using the limiting dilution method to obtain hybridoma cell lines with a positive rate of 100%. The ELISA identification results are shown in Table 2.

[0096] Table 2 Results of ELISA identification of hybridoma cell lines secreting rabies virus antibodies

[0097]

[0098]

[0099] The hybridoma cell lines secreting anti-rabies virus monoclonal antibodies harvested in the previous step were further identified by western-bolt method, wherein the hybridoma cell lines secreting anti-RV G protein monoclonal antibodies and anti-RV N protein monoclonal antibodies were identified. The specific steps of western-bolt are:

[0100] 80 μl of inactivated rabies virus solution with a protein concentration of 1 mg / ml was mixed with 20 μl of 4× SDS-PAGE protein loading buffer and boiled in boiling water for 8 min. The mixture was then centrifuged at 5000 rpm for 3 min, and 5 μl of the supernatant was taken and subjected to SDS-PAGE polyacrylamide gel electrophoresis together with a protein marker.

[0101] After the electrophoresis is finished, take out the gel, cut a nitrocellulose membrane and six filter papers of corresponding size, soak the gel, membrane, filter paper and sponge in transfer buffer, install them in the order of "sponge-three-layer filter paper-gel-nitrocellulose membrane-three-layer filter paper-sponge", put them into the transfer device, and transfer them at a current of 90mA and a transfer time of 90min;

[0102] After the transfer was completed, the membrane was placed in a blocking solution (10 mmol / L PBS buffer containing 5 wt% skim milk powder) and blocked overnight at 4°C;

[0103] The membrane was removed from the blocking solution and washed three times with PBST solution, each time for 8 minutes. The membrane was then placed in hybridoma cell supernatant diluted 1:1000 with 1% skim milk powder 10mmol / L PBS buffer, incubated at room temperature for 1 hour, and then washed three times with PBST, each time for 10 minutes;

[0104] The membrane was placed in a 50,000-fold dilution of Hrp-labeled goat anti-mouse secondary antibody solution in PBS buffer containing 1% skimmed milk powder (10 mmol / L). After incubation at room temperature for h, the membrane was washed three times with PBST, each time for 10 min.

[0105] The membrane was immersed in ECL chemiluminescent solution for 2-3 minutes, and then the analysis results were observed in the Western Blot imaging system.

[0106] 2. Experimental results

[0107] Western Blot results Figure 1 and Figure 2 As shown, the results showed that RV22 showed a bright band in the binding reaction with the glycoprotein near the 58KD position, and RV26 showed a bright band in the binding reaction with the nucleoprotein near the 50KD position. It indicates that the hybridoma cell line YB-RV22 (deposited in China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, the deposit number is CCTCC NO: C202220, the deposit date is January 19, 2022) that stably secretes anti-RVG protein (rabies virus glycoprotein) antibodies, and the hybridoma cell line YB-RV26 (deposited in China Center for Type Culture Collection, the deposit address is: Wuhan University, Wuhan, China, the deposit number is CCTCC NO: C2022283, the deposit date is September 21, 2022) that secretes anti-RVN protein (rabies virus nucleoprotein) antibodies have been obtained.

[0108] IV. Obtaining anti-RV G protein monoclonal antibodies and anti-RV N protein monoclonal antibodies by in vitro culture method:

[0109] 1. Experimental methods

[0110] The hybridoma cells (YB-RV22 and YB-RV26) obtained by culture in a cell fermentation device were centrifuged to remove cells and their debris, and the cell culture supernatant was collected. The anti-RV G protein monoclonal antibody and the anti-RV N protein monoclonal antibody were separated and purified from the cell culture supernatant using a Protein A-Sepharose affinity chromatography column. Subsequently, the subtypes of the obtained anti-RV G protein monoclonal antibody and anti-RV N protein monoclonal antibody were identified using a mouse monoclonal antibody subtype identification ELISA kit (C060101-L) according to the instructions.

[0111] 2. Experimental results

[0112] It has been identified that the hybridoma cell line YB-RV22 against RV G protein (deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: C202220, and a deposit date of January 19, 2022), and the hybridoma cell line YB-RV26 against RV N protein antibody (deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: C2022283, and a deposit date of September 21, 2022), each secretes monoclonal antibodies of the IgG1 type, and the light chains are both κ chains.

[0113] Example 2 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0114] Schematic diagram of the principle of the non-free nucleoprotein detection method for rabies vaccine samples Figure 3 As shown, the specific steps include:

[0115] S1: Preparation of solid phase monoclonal antibody materials

[0116] The anti-RV G protein monoclonal antibody prepared in Example 1 (secreted by the hybridoma cell line YB-RV22 deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: C202220 and a deposit date of January 19, 2022) was used as the primary antibody, and a Tris-HCl buffer with a pH of 7.2 and a concentration of 50 mmol / L was used as the primary antibody diluent to dilute the primary antibody to a concentration of 3 μg / mL, and added to a 96-well ELISA plate at 100 μL / well, and the plate was sealed with a sealing film and incubated at 4°C overnight; then a Tris-HCl buffer containing 0.84 wt% NaCl, 0.01 wt% NaN3, 0.06 vol% Tween-20, pH = 7.8, and a concentration of 50 mmol / L was used as a washing solution, and the ELISA plate was washed 5 times with 300 μL / well of the washing solution, and the detection plate was patted dry for the last time to obtain a coated ELISA plate.

[0117] The blocking solution (1g BSA was weighed and fully dissolved in 95g Na2CO3-NaHCO3 buffer with a pH of 9.6 and a concentration of 50mmoL / L, and then weighed to 100g with deionized water to prepare the blocking solution) was added to the coated ELISA plate at 300μL / well and blocked at 37°C for 2h; then the plate was washed 5 times with 300μL / well of washing solution, and the detection plate was patted dry for the last time and stored at below -20°C to obtain a blocked ELISA plate, which is the solid phase monoclonal antibody material.

[0118] S2:Eu 3+ Preparation of labeled anti-RV N protein monoclonal antibody

[0119] A Na2CO3-NaHCO3 buffer solution containing 155mmol / L NaCl, pH=8.5, and a concentration of 50mmol / L was used as a labeling solution; 1mL of labeling solution was added to 1mg of anti-RV N protein monoclonal antibody (secreted by hybridoma cell line YB-RV22 deposited in China Center for Type Culture Collection, with a deposit number of CCTCCNO: C2022283 and a deposit date of September 21, 2022), and then 0.2mg of SCN-Ph-DTTA-Eu 3+ The mixture was stirred gently at room temperature for 18 h; the mixture was then purified using a Sephadex G50 column to obtain Eu 3+ The complex-labeled anti-RV N protein monoclonal antibody, i.e., the secondary antibody, is stored at a temperature below -20°C.

[0120] S3: Preparation of ELISA plate to be tested

[0121] Add the sample to be tested: use Tris-HCl buffer containing 0.05wt% NaN3, 0.2wt% bovine serum albumin, pH 7.8, and concentration of 50mmol / L as the protein sample diluent, add the serially diluted rabies vaccine reference solution (0EU / ml, 2.5EU / ml, 10EU / ml, 100EU / ml, 500EU / ml and 1000EU / ml) and the sample solution to be tested to the closed ELISA plate at 100μL / well, seal the plate with a sealing film, and incubate at room temperature for 1h with shaking; wash the ELISA plate with 300μL / well of washing solution for 4 times, and pat the test plate dry for the last time to obtain the ELISA plate containing the sample to be tested.

[0122] Adding secondary antibody: using Tris-HCl buffer containing 0.03wt% BSA, 0.05vol% Tween-20, 0.05wt% NaN3, pH=7.8, and a concentration of 50mmol / L as the secondary antibody diluent, dilute the concentration of the secondary antibody to 200μg / mL, add the diluted secondary antibody to the ELISA plate containing the sample to be tested at 100μL / well, seal the plate with a sealing film, and incubate at room temperature with shaking for 1h; wash the ELISA plate 6 times with 300μL / well of washing solution, and pat the test plate dry for the last time to obtain an ELISA plate containing the secondary antibody.

[0123] Add enhancement solution: Use 100 mmol / L acetic acid-phthalic acid buffer solution containing 50 μmol / L TOPO, 15 μmol / L β-NAT, 0.1 vol% Triton X-100, pH = 7.8, and a concentration of 100 μL as the enhancement solution. Add 100 μL / well to the ELISA plate containing the secondary antibody, incubate at room temperature with shaking for 5 minutes, and obtain the ELISA plate to be tested.

[0124] S4: Determination of non-free nuclear protein content in the sample to be tested

[0125] Using semi-automatic Victor3 TM The 1420 multi-label detector detects the ELISA plate to be tested, measures the fluorescence signal values ​​of a series of concentrations of rabies vaccine calibrator solutions, and draws a standard curve by double logarithmic fitting of the concentration and the fluorescence signal value.

[0126] According to the standard curve, calculate the content of non-free nuclear protein in the sample to be tested. The standard curve is shown in Figure 4 The results showed that the linear range could reach 1000Eu / ml, R 2 It is 0.9995, indicating a good correlation.

[0127] Comparative Example 1: A method for detecting non-free nucleoprotein in rabies vaccine samples

[0128] Compared with Example 2, only the blocking solution in step S1 is changed. The preparation method is as follows: 0.8 g BSA is weighed and fully dissolved in 95 g of Na2CO3-NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and then weighed to 100 g with deionized water to prepare the blocking solution.

[0129] Example 3 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0130] Compared with Example 2, only the blocking solution in step S1 is changed. The preparation method is as follows: 1.2 g BSA is weighed and fully dissolved in 95 g Na2CO3-NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and the weight is adjusted to 100 g with deionized water to prepare the blocking solution.

[0131] Example 4 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0132] Compared with Example 2, only the blocking solution in step S1 is changed. The preparation method is as follows: 1.5 g BSA is weighed and fully dissolved in 95 g Na2CO3-NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and then weighed to 100 g with deionized water to prepare the blocking solution.

[0133] Comparative Example 2: A method for detecting non-free nucleoprotein in rabies vaccine samples

[0134] Compared with Example 2, only the blocking solution in step S1 is changed. The preparation method is as follows: 1.2 g of skimmed milk powder is weighed and fully dissolved in 95 g of Na2CO3-NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and then weighed to 100 g with deionized water to prepare the blocking solution.

[0135] Comparative Example 3: A method for detecting non-free nucleoprotein in rabies vaccine samples

[0136] Compared with Example 2, only the blocking solution in step S1 is changed. The preparation method is as follows: 1.2 g of horse serum albumin is weighed and fully dissolved in 95 g of Na2CO3-NaHCO3 buffer with a pH of 9.6 and a concentration of 50 mmol / L, and then weighed to 100 g with deionized water to prepare a blocking solution.

[0137] Comparative Example 4: A method for detecting non-free nucleoprotein in rabies vaccine samples

[0138] Compared with Example 2, only SCN-Ph-DTTA-Eu 3+ The dosage is 0.05mg.

[0139] Example 5 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0140] Compared with Example 2, only SCN-Ph-DTTA-Eu 3+ The dosage is 0.1mg.

[0141] Example 6 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0142] Compared with Example 2, only SCN-Ph-DTTA-Eu 3+ The dosage is 0.3mg.

[0143] Comparative Example 5: A method for detecting non-free nucleoprotein in rabies vaccine samples

[0144] Compared with Example 2, only the concentration of the primary antibody added to the ELISA plate after dilution in step S1 was changed from 3 μg / mL to 2 μg / mL.

[0145] Example 7 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0146] Compared with Example 2, only the concentration of the primary antibody added to the ELISA plate after dilution in step S1 was changed from 3 μg / mL to 4 μg / mL.

[0147] Example 8 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0148] Compared with Example 2, only the concentration of the primary antibody added to the ELISA plate after dilution in step S1 was changed from 3 μg / mL to 5 μg / mL.

[0149] Comparative Example 6: A method for detecting non-free nucleoprotein in rabies vaccine samples

[0150] Compared with Example 2, only the concentration of the secondary antibody after dilution in step S3 was changed from 2 μg / mL to 1.6 μg / mL.

[0151] Example 9 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0152] Compared with Example 2, only the concentration of the secondary antibody after dilution in step S3 was changed from 2 μg / mL to 1.8 μg / mL.

[0153] Example 10 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0154] Compared with Example 2, only the concentration of the secondary antibody after dilution in step S3 was changed from 2 μg / mL to 2.2 μg / mL.

[0155] Example 11 A method for detecting non-free nucleoprotein in rabies vaccine samples

[0156] Compared with Example 2, only the concentration of the secondary antibody after dilution in step S3 was changed from 2 μg / mL to 2.4 μg / mL.

[0157] Example 12 A kit for detecting non-free nucleoprotein in rabies vaccine samples

[0158] 1. Composition

[0159] The monoclonal antibody against rabies virus glycoprotein is secreted by the hybridoma cell line YB-RV22, which is deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: C202220 and the deposit date: January 19, 2022;

[0160] The monoclonal antibody against rabies virus nucleoprotein is secreted by the hybridoma cell line YB-RV26, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: C2022283 and the deposit date is September 21, 2022;

[0161] It also contains blocking solution, enhancement solution, washing solution, labeling solution, protein diluent and secondary antibody diluent;

[0162] The blocking solution is a BSA solution with a mass concentration of 1%;

[0163] The enhancement solution is an acetic acid-phthalic acid buffer solution containing 50 μmol / L TOPO, 15 μmol / L β-NAT, 0.1 vol% Triton X-100, pH=7.8, and a concentration of 100 mmol / L;

[0164] The washing solution is a Tris-HCl buffer containing 0.84 wt % NaCl, 0.01 wt % NaN3, 0.06 vol % Tween-20, pH=7.8, and a concentration of 50 mmol / L.

[0165] The labeling solution is a Na2CO3-NaHCO3 buffer solution containing 155mmol / L NaCl, pH=8.5, and a concentration of 50mmol / L;

[0166] The sample diluent is a Tris-HCl buffer containing 0.05wt% NaN3, 0.2wt% bovine serum albumin, pH 7.8, and a concentration of 50mmol / L;

[0167] The secondary antibody diluent contains 0.03 wt % BSA, 0.05 vol % Tween-20, 0.05 wt % NaN3, pH=7.8, and a Tris-HCl buffer with a concentration of 50 mmol / L.

[0168] 2. Usage

[0169] Anti-rabies virus glycoprotein monoclonal antibody (3 μg / mL, 100 μL / well) was coated on an ELISA plate, the ELISA plate was washed 5 times with 300 μL / well washing solution, patted dry, and then blocked with a blocking solution, the ELISA plate was washed 5 times with 300 μL / well washing solution, patted dry, to obtain a solid phase monoclonal antibody material;

[0170] 1 mg of monoclonal antibody against rabies virus nucleoprotein, 1 mL of labeling solution and 0.2 mg of SCN-Ph-DTTA-Eu 3+ Mix, shake gently at room temperature, react for 18 h, label, and purify the mixture with Sephadex G50 column to obtain Eu 3+ The complex is labeled with a detection antibody to prepare a secondary antibody;

[0171] The solid phase monoclonal antibody material was added with the sample to be tested (100 μL / well) and the secondary antibody (200 μg / mL, 100 μL / well), and the plate was sealed with a sealing film, and then incubated at room temperature with shaking for 1 hour; the ELISA plate was washed 6 times with 300 μL / well of washing solution, and the detection plate was patted dry for the last time to obtain an ELISA plate containing the secondary antibody; then, the enhancement solution (100 μL / well) was added and incubated at room temperature with shaking for 5 minutes to obtain an ELISA plate to be tested; wherein the sample to be tested was diluted with a sample diluent, and the secondary antibody was diluted with a secondary antibody diluent;

[0172] The semi-automatic Victor3TM 1420 multi-label detector was used to perform fluorescence measurement on the ELISA plate to be tested, and the reaction curve was drawn based on the test results of the calibration solution with different concentrations to analyze the content of non-free nuclear protein in the sample to be tested.

[0173] Application Example 1: Accuracy and Precision of Non-free Nucleoprotein Detection Method in Rabies Vaccine Samples

[0174] 1. Experimental Methods

[0175] A rabies vaccine reference solution with a concentration of 5EU / mL was used as the sample to be tested. The non-free nucleoprotein content in the sample to be tested was detected using the detection methods provided in Examples 2 to 11 and Comparative Examples 1 to 6. The determination was repeated 6 times, and statistical processing was performed to calculate the recovery rate and coefficient of variation of the detection method. The accuracy and precision of the detection methods provided in Examples 1-16 and the comparative examples were compared.

[0176] Wherein, recovery rate = (average value of measured concentration / theoretical concentration) × 100%; coefficient of variation = (standard deviation / average value of measured concentration) × 100%.

[0177] 2. Experimental Results

[0178] The results are shown in Table 3.

[0179] Table 3 Accuracy and precision of the detection method when testing samples of the same concentration

[0180]

[0181]

[0182] Combined with Table 3, by comparing the test results of Examples 2 to 11 with Comparative Examples 1 to 6, using the anti-RV G protein monoclonal antibody, anti-RV N protein monoclonal antibody and time-resolved fluorescence immunoassay method prepared in Example 1, when repeatedly testing the sample to be tested, the recovery rate of the test result of the present invention can be controlled within the range of 90% to 100%, and the coefficient of variation is less than 10%. The above test results show that the time-resolved fluorescence immunoassay method for the non-free nucleoprotein content of rabies virus and / or rabies vaccine provided by the present invention can achieve higher accuracy and precision.

[0183] Specifically, by comparing the test results of Examples 2 to 4 with Comparative Example 1, it can be seen that when the concentration of the BSA solution in the blocking solution is controlled within the range of 1 to 1.5 wt%, the accuracy and precision of the test results can be further improved. Therefore, the present invention controls the concentration of the BSA solution within the above range.

[0184] By comparing the test results of Examples 2 to 4 with Comparative Examples 2 and 3, compared with using skimmed milk powder solution or horse serum albumin solution, using BSA solution as the blocking solution can further improve the accuracy and precision of the test results. Therefore, BSA is selected as the active ingredient in the blocking solution.

[0185] By comparing the test results of Examples 2, 5 to 6 and Comparative Example 4, it can be seen that when the lanthanide element europium ion complex SCN-Ph-DTTA-Eu 3+ When the weight ratio of lanthanide ions to anti-RV N protein monoclonal antibodies is within the range of (0.1-0.3):1, the accuracy and precision of the detection results can be significantly improved. Therefore, the present invention controls the weight ratio of lanthanide ions to anti-RV N protein monoclonal antibodies within the above range.

[0186] By comparing the test results of Examples 2, 7 to 8 and Comparative Example 5, it can be seen that when the coating concentration of the primary antibody is controlled within the range of 3 to 5 μg / mL, the accuracy and precision of the test results can be further improved. Therefore, the present invention controls the concentration of the primary antibody within the above range.

[0187] By comparing the test results of Examples 2, 9 to 11 and Comparative Example 6, it can be seen that when the concentration of the secondary antibody is controlled within the range of 1.8 to 2.2 μg / mL, the accuracy and precision of the test results can be further improved. Therefore, the present invention controls the concentration of the secondary antibody within the above range.

[0188] Application Example 2: Non-free nucleoprotein detection method for rabies vaccine samples to detect samples with different particle integrity states

[0189] 1. Experimental Methods

[0190] A rabies vaccine reference solution with a concentration of 5 EU / mL was used as the original sample. The samples were treated with different virus particle disruption methods (ultrasonic disruption and virus lysis solution, see Table 4). The non-free nucleoprotein content in the treated samples was measured three times using the method of Example 2, and statistical processing was performed to calculate the detection value of the sample.

[0191] 2. Experimental Results

[0192] The results are shown in Table 4.

[0193] Table 4 Detection and analysis results of samples with different virus integrity status:

[0194] Sample No. Destruction method Average concentration of the assay (EU / mL) 1 300w ultrasonic treatment for 30s 5.21 2 300w ultrasonic treatment 60s 6.21 3 300w ultrasonic treatment 300s 14.73 4 300w ultrasonic treatment 600s 20.1 5 20% concentration lysis buffer treatment 18.37 6 5% concentration lysis buffer treatment 7.65

[0195] By comparing the test results of samples 1 to 4, the longer the ultrasound time, the worse the integrity of the virus particles and the more non-free nucleoprotein detected.

[0196] By comparing samples 5 to 7 after adding different concentrations of lysis buffer, the integrity of rabies virus particles decreased as the concentration of lysis buffer increased, and the detection amount of non-free nucleoprotein increased.

[0197] In summary, the detection method can analyze and evaluate the integrity of rabies virus particles in rabies vaccine samples by detecting the non-free nucleoprotein content in the sample. The method for evaluating the integrity of rabies virus particles by detecting non-free nucleoprotein disclosed in the present invention can be used to monitor the effects of different process treatments on the integrity of rabies virus particles during the production of rabies vaccines. This method overcomes the shortcomings of high requirements for electron microscope equipment and the inability to carry out the work by itself. It can quickly and easily complete the evaluation of particle integrity in laboratories related to vaccine quality control, effectively guarantee the quality of the vaccine and assist in improving related processes.

[0198] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A hybridoma cell line composition, characterized in that: Including a hybridoma cell line that secretes monoclonal antibodies against rabies virus glycoprotein and a hybridoma cell line that secretes monoclonal antibodies against rabies virus nucleoprotein: the hybridoma cell line that secretes monoclonal antibodies against rabies virus glycoprotein is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: C202220, and a deposit date of January 19, 2022; the hybridoma cell line that secretes monoclonal antibodies against rabies virus nucleoprotein is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: C2022283, and a deposit date of September 21, 2022.

2. A monoclonal antibody composition, characterized in that: include: Monoclonal antibodies against rabies virus glycoprotein and monoclonal antibodies against rabies virus nucleoprotein, wherein the monoclonal antibodies against rabies virus glycoprotein are monoclonal antibodies secreted by the hybridoma cell line that secretes the monoclonal antibodies against rabies virus glycoprotein in the hybridoma cell line composition of claim 1, and the monoclonal antibodies against rabies virus nucleoprotein are monoclonal antibodies secreted by the hybridoma cell line that secretes the monoclonal antibodies against rabies virus nucleoprotein in the hybridoma cell line composition of claim 1.

3. Use of the hybridoma cell line composition according to claim 1 or the monoclonal antibody composition according to claim 2 in the preparation of a kit for detecting the content of non-free nucleoprotein of rabies virus and / or rabies vaccine.

4. Use of the hybridoma cell line composition according to claim 1 or the monoclonal antibody composition according to claim 2 in the preparation of a kit for detecting the integrity of rabies virus particles.

5. An immunoassay kit for detecting the content of non-free nucleoprotein of rabies virus and / or rabies vaccine, and / or the integrity of rabies virus particles, characterized in that The immunoassay kit comprises the monoclonal antibody composition of claim 2, wherein the monoclonal antibody against rabies virus glycoprotein in the monoclonal antibody composition of claim 2 is used as a primary antibody; and the monoclonal antibody against rabies virus nucleoprotein in the monoclonal antibody composition of claim 2 labeled with a lanthanide ion complex is used as a secondary antibody.

6. A method for detecting the content of non-free nucleoprotein of rabies virus and / or rabies vaccine, and / or the integrity of rabies virus particles, characterized in that: The following steps are involved: The monoclonal antibody against rabies virus glycoprotein in the monoclonal antibody composition of claim 2 is coated on an ELISA plate, and blocked with a blocking solution to obtain a solid phase monoclonal antibody material; Using a lanthanide ion complex to label the monoclonal antibody against rabies virus nucleoprotein in the monoclonal antibody composition of claim 2 to prepare a secondary antibody; The sample to be tested and the secondary antibody are added to the solid phase monoclonal antibody material, and then an enhancement solution is added for incubation to obtain an ELISA plate to be tested; The ELISA plate to be tested is subjected to fluorescence measurement, and the obtained fluorescence signal is substituted into the standard curve equation obtained by fitting according to the concentration of the calibrator and the signal data to calculate the content of non-free nuclear protein in the sample to be tested.

7. The detection method according to claim 6, characterized in that: The lanthanide ion complex is N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid-Eu 3+ The weight ratio of the monoclonal antibody against rabies virus nucleoprotein to the monoclonal antibody composition of claim 2 is (0.1-0.3):

1.

8. The detection method according to claim 6, characterized in that: The coating concentration of the primary antibody is 3-5 μg / mL; the working concentration of the secondary antibody is 1.8-2.2 μg / mL.

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